<i>Brucella abortus</i> is capable of resisting the microbicidal mechanisms of phagocytic cells and growing within phagocytic cells, usually macrophages. <I>B. abortus</i>, like several other intracellular bacteria responds to the hostile environment in macrophages by producing heat shock proteins (HSPs) which are induced by environmental stresses. Bacterial HSPs are very immunogenic, eliciting both cellular and humoral immune responses in the infected host. The significance of host cellular and protective immune responses directed against these proteins is currently unresolved. Baculovirus recombinants were generated in <i>Sf9</i> insect cells for <i>B. abortus</i> HSPs and the protein expression was optimized. Humoral (Western blot), cell mediated (CMI, IFN-g- release by splenocytes, and CD3+CD4+, CD3+CD8+ T cell/ total splenocytes ratios) and protective immune responses of BALB/c mice (challenge with virulent <i>B. abortus</i> 2308) against these recombinants, against <i>B. abortus</i> superoxide dismutase (SOD) and ribosomal L7/L12 proteins, inoculated alone or in various combinations with complete Freund's, Ribi and recombinant IL-12 as adjuvants, were analyzed. Vaccinia virus-GroEL recombinant as priming immunogen, followed by baculovirus-GroEL-Ribi booster, was explored. Androstenediol, an immune up-regulator, was tested for its ability to induce resistance against challenge. None of the mice inoculated with individual, divalent or trivalent HSP-expressing <i>Sf9</i> cells combined with Freund's were protected against challenge and the <i>Sf9</i> cell-induced response masked the recombinant protein-specific CMI responses. Recombinant HSPs were purified and combined with Ribi. Although significant IFN-g release was induced by immunization with the HtrA-Ribi combination, no mice were protected against challenge. Priming with vaccinia virus-GroEl recombinant and boosting with purified baculovirus-GroEL protein-Ribi combination did not induce protection. Androstenediol did not enhance in vivo resistance to challenge. IL-12 alone did not activate splenocytes but induced significant IFN-g release in mice when combined with killed <i>B. abortu</i>s RB51 vaccine, purified recombinant HtrA or purified SOD proteins, or L7/L12 expressing <i>Escherichia coli</i> cells. Significant protection was induced by SOD combined with IL-12. No correlation was seen between IFN-g release by splenocytes and protection against challenge in the SOD/IL-12-immunized mice. The results suggest that <i>B. abortus</i> HSPs are not highly immunogenic in mice and though various immune responses may be induced by one or another HSPs, protective immune response, unfortunately, is not among them. The results of this study reflect the difficulties in experimenting with immune responses against single or a limited number of recombinant <i>B. abortus</i> proteins. This is particularly true when the task includes induction of a protective immune response and finding significant correlation between different types of immune response assays. / Ph. D.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Le Laboratoire de génie tissulaire est reconnu pour ses réalisations en ce domaine. Le principal défi soulevé par cette approche est le choix de la matrice des tissus reconstruits. Mes travaux ont consisté à établir une technologie de synthèse de collagène humain recombinant à des fins expérimentales et cliniques. Ce collagène sera utilisé éventuellement pour produire des substituts du ligament croisé antérieur (LCA) du genou, par génie tissulaire. Ces substituts ligamentaires pourraient être une alternative de remplacement des LCA rupturés. Le Dr. Nazrul Islam a conceptualisé une stratégie moléculaire pour construire un plasmide incluant les gènes codant pour les deux chaînes du collagène humain de type 1 et les deux sous-unités de l'enzyme prolyl-4-hydroxylase. Des cellules d'insecte ont été transfectées avec ce vecteur, en exploitant le système de bacul ovi rus, pour synthétiser le collagène recombinant. J'ai participé à chaque étape et à la mise au point des protocoles optimisé à grande échelle de cette nouvelle technologie, pour ensuite purifier le collagène et le caractériser biochimiquement. Mes superviseurs et moi-même considérons que ces travaux ont réussi et que bientôt, des substituts ligamentaires humains seront greffés pour évaluer leur intégration dans une articulation du genou in vivo.

L'utilisation thérapeutique de glycoprotéines recombinantes produites dans le système baculovirus-cellules d'insectes reste limitée par le potentiel de glycosylation de ces cellules, qui produisent essentiellement des structures O- et N-glycanniques courtes. Notre travail s'inscrit donc dans un effort global d'"humanisation" de la glycosylation des protéines produites dans les cellules de Lépidoptères.<br />Nous nous sommes d'abord attachés à comprendre la voie de maturation des N-glycannes dans des cellules Sf9. L'utilisation d'inhibiteurs de la maturation ou du trafic intracellulaire nous a permis d'identifier des intermédiaires clés et de confirmer l'hypothèse que la maturation des N-glycannes dans les cellules d'insectes et de mammifères suivent un chemin métabolique parallèle jusqu'à la formation de l'espèce GlcNAcMan3[Fuc]GlcNAc2. Chez les insectes, cette structure est ensuite substrat d'une β-N-acétylglucosaminidase qui produit l'espèce finale Man3[Fuc]GlcNAc2.<br />Cette voie de maturation peut néanmoins être déviée vers la synthèse de N-glycannes de type complexe par l'addition de glycosyltransférases absentes : ainsi, l'expression d'une β1,4-galactosyltransférase permet la synthèse de l'espèce GalGlcNAcMan3[Fuc]GlcNAc2.<br />Notre intérêt s'est ensuite porté sur l'ingénierie de la sialylation dans les cellules d'insectes, compliquée par l'absence du donneur d'acides sialiques, le CMP-Neu5Ac. Notre stratégie a été d'exprimer la trans-sialidase de Trypanosoma cruzi sur la membrane plasmique des cellules, afin qu'elle puisse sialyler les glycoprotéines sécrétées en utilisant des donneurs du milieu. La construction exprimée grâce à un vecteur baculovirus code une enzyme active, dont une partie est retrouvée sur la membrane plasmique et sur l'enveloppe des particules virales, tandis qu'une partie, croissante avec l'infection, est soluble. Néanmoins, le système permet une sialylation quantitative d'accepteurs exogènes.<br />Notre étude contribue à montrer que l'ingénierie de la glycosylation dans le système baculovirus-cellules d'insectes est envisageable. Pour résoudre le problème de la sialylation, la trans-sialidase est une alternative possible aux sialyltransférases.

Recentemente, vários arbovírus têm acometido a população de países emergentes ocasionando sérios problemas de saúde pública, como as doenças causadas pelos vírus da dengue, Chikungunya, Zika e febre amarela. Um vírus emergente e já circulante no Brasil, chamado Mayaro (MAYV), do mesmo gênero do Chikungunya (Alphavirus), possui potencial prejudicial semelhante a esses já estabelecidos. Seu vetor de transmissão é o mosquito do gênero Haemagogus, característico de regiões isoladas, principalmente florestas. Entretanto, estudos demonstraram que o Aedes aegypti é um competente vetor desse agente, o que possibilita sua disseminação em regiões urbanas. O presente trabalho avaliou a expressão das proteínas estruturais do vírus Mayaro (E1, E2, E3, C e 6K), utilizando dois sistemas de expressão distintos, um baseado na levedura Pichia pastoris, e outro derivado de Baculovírus (BEVS). Essa estratégia foi estabelecida para que a expressão dessas proteínas promova a formação de partículas semelhantes ao vírus (virus like particles), estruturas multiprotéicas que mimetizam a conformação de uma partícula viral podendo ser utilizada como um candidato vacinal. O trabalho evidenciou a correta obtenção de organismos recombinantes em ambos os sistemas, com a avaliação da expressão sendo feita com técnicas de dot blot, western blot e imunofluorescência indireta (IFI). Com o sistema baculovírus, foram avaliadas as linhagens Sf-9 e Hi-5, sendo evidenciada a expressão de proteínas do MAYV em ambas, utilizando MOI 10 e tempos pós-infecção de 96 e 72 h, respectivamente. A correta expressão das proteínas de MAYV também foi evidenciada com a levedura Pichia pastoris, com cultivo a 30 &deg;C e tempo de análise 48 h após indução. A geração de VLPs foi avaliada em amostras de sobrenadantes de ambos os sistemasapós a concentração por ultracentrifugação em gradiente de iodixanol, e análise por microscopia eletrônica de transmissão, sendo observadas nos dois sistemas com tamanhos variando entre 30-60 nm. Os resultados desse projeto podem gerar ferramentas importantes no desenvolvimento de kits diagnósticos e métodos vacinais contra o MAYV. / Recently, several arboviruses have affected emerging countries causing serious public health problems, such as diseases caused by dengue viruses, Chikungunya, Zika and yellow fever. An emerging and already circulating virus in Brazil, called Mayaro (MAYV), of the same genus of Chikungunya (Alphavirus), has harmful potential similar to those already established. Its transmission vector is the mosquito of the genus Haemagogus, characteristic of isolated regions, mainly forests. However, studies have demonstrated that Aedes aegypti is a competent vector of this agent, which would allow its dissemination in urban areas. The present work evaluated the expression of the structural proteins of the Mayaro virus (E1, E2, E3, C and 6K) using two distinct expression systems, one based on the yeast Pichia pastoris and another derived from Baculovirus (BEVS). The strategy of expressing structural proteins has been established so that the expression of these proteins promotes the formation of viruslike particles or VLPs, multiprotein structures that mimic the conformation of a viral particle and can be used as a vaccine candidate. The work evidenced the correct obtaining of recombinant organisms in both systems, with the evaluation of the expression being done with dot blot, western blot and indirect immunofluorescence (IFI) techniques. With the baculovirus system, the Sf-9 and Hi-5 strains were evaluated, and the expression of the MAYV proteins was evidenced in both, using MOI 10 and the time of post-infection analysis of 96 and 72 h, respectively. Correct expression of MAYV proteins was also evidenced with yeast Pichia pastoris, with culture at 30 &deg; C and analysis time 48 h after induction. The generation of VLPs was evaluated in both supernatants after concentration by iodixanol gradient ultracentrifugation and transmission electron microscopy analysis, being observed in both systems with sizes ranging from 30-60 nm. The results of this project can generate important tools in the development of diagnostic kits and vaccine methods against the MAYV.

Submitted by Verena Bastos (verena@uefs.br) on 2015-09-21T13:18:11Z No. of bitstreams: 1 DISSERTACAO FINAL ALINE THOMAZ.pdf: 1680438 bytes, checksum: 85bf2d7886a394df745618b6fd50d435 (MD5) / Made available in DSpace on 2015-09-21T13:18:11Z (GMT). No. of bitstreams: 1 DISSERTACAO FINAL ALINE THOMAZ.pdf: 1680438 bytes, checksum: 85bf2d7886a394df745618b6fd50d435 (MD5) Previous issue date: 2015-09-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The vitamin D receptor (VDR) is a cytoplasmic transcription factor and when activated by its ligand, translocates to the nucleus and interacts with DNA in the promoter regions with which has affinity, acting as a modulator of gene transcription and thereby producing multiple biological effects. Its main activities are the regulation and maintenance of plasma levels of calcium and phosphorus, as well as presenting immunomodulatory activities, such as the suppression of T cell activation, formation of secretion patterns of cytokines, modulation of proliferation and interference in the apoptosis. So this receptor is an important target for drugs that can help in the discovery of new immunomodulators. The present work had the objective to produce the recombinant vitamin D receptor in its soluble form for conducting future assays of drug screening of potential immunomodulators and drug-receptor interaction studies. For this, we used initially Escherichia coli expression system transformed with the plasmid HS_VDR_EC1-PQE T7, but it was only possible to obtain the protein in the insoluble fraction, even varying the temperature, time of induction and IPTG concentration. In an attempt to obtain soluble VDR, we used a eukaryotic expression system in insect cells using the baculovirus as a vector. It was built a vector, pFASTBacHT_VDR, which had the sequence of this protein cloned from pCMX.VDR. From there, it was possible to obtain recombinant bacmids used in transfection of insect cells, generating recombinant baculovirus, to then proceed with the expression of VDR. / O receptor de vitamina D (VDR) ? um fator de transcri??o g?nica citoplasm?tico e, quando ativado pelo seu ligante, transloca-se para o n?cleo e interage com regi?es promotoras no DNA com a qual apresenta afinidade, atuando como fator modulador da transcri??o g?nica e assim produzindo m?ltiplos efeitos biol?gicos. Suas principais atividades s?o a regula??o e a manuten??o dos n?veis plasm?ticos de c?lcio e f?sforo, e apresenta atividades imunomoduladoras, como a supress?o da ativa??o de c?lulas T, forma??o de padr?es de secre??o de citocinas, a modula??o da prolifera??o e interfer?ncia na apoptose. Sendo assim, esse receptor representa um importante alvo de drogas que pode contribuir na descoberta de novos f?rmacos com a??o imunomoduladora. O presente trabalho teve, como objetivo, produzir o VDR recombinante na forma sol?vel para a realiza??o de futuros ensaios de triagem de potenciais drogas com a??o imunomoduladora e estudos de intera??o droga-receptor. Para isso, foi utilizado, inicialmente, o sistema de express?o em Escherichia coli, utilizando o plasm?deo HS_VDR_EC1-PQE T7, por?m s? foi poss?vel obter a prote?na na fra??o insol?vel, mesmo variando a temperatura, o tempo de indu??o e a concentra??o de IPTG. Na tentativa de obter o VDR sol?vel, foi utilizado um sistema de express?o eucari?tico em c?lulas de inseto, utilizando como vetor o baculov?rus. Foi constru?do um vetor pFASTBacHT_VDR, o qual teve a sequ?ncia desta prote?na clonada a partir do pCMX.VDR. A partir da?, foi poss?vel obter bacm?deos recombinantes, utilizados na transfec??o de c?lulas de inseto, gerando baculov?rus recombinantes, para, ent?o, seguir com a express?o do VDR.

Le VHB est un problème majeur puisque les 350 millions de porteurs chroniques existant ont un risque accru de développer une cirrhose ou un carcinome hépatocellulaire. Compte tenu du manque de système d'étude du VHB in vitro qui soit facile d'accès et pleinement satisfaisant, l'objectif était d'améliorer l'un de ceux qui utilisent des baculovirus VHB recombinants pour délivrer le génome VHB dans des cellules hépatocytaires. La pertinence de ce système pour réaliser des tests phénotypiques et étudier le « fitness » des mutants résistants aux antiviraux a ensuite été démontrée. Enfin, l'utilisation de ces baculovirus VHB recombinants dans des cellules HepaRG a permis de mettre en évidence une réponse IFN efficace de l'hépatocyte suite à la synthèse des protéines du VHB. Ceci constitue une donnée nouvelle dans l'étude des interactions virus/cellules puisque le VHB était considéré jusqu'à présent comme un virus silencieux vis-à-vis de la réponse innée cellulaire. L'ensemble de ces résultats a des implications importantes dans la compréhension des mécanismes de persistance du VHB et le développement de nouveaux modèles cellulaires d'infection.

Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive disorder caused by a defect in alpha-N-acetylglucosaminidase (NAGLU), a lysosomal enzyme involved in the degradation of heparan sulphate. Dysfunctional NAGLU gives rise to a clinical phenotype of severe and progressive mental retardation, often accompanied by hyperactivity and aggressive behaviour. At present, there is no effective treatment for MPS IIIB. However, cloning of the human NAGLU cDNA has made the potential production of human recombinant enzyme for use in enzyme replacement therapy (ERT) a viable option. The work outlined herein focuses on attempts to produce human recombinant NAGLU (rNAGLU) using both yeast and insect cell based expression systems; with the major focus on yeast based expression. Use of a humanized yeast strain, codon optimisation of a portion of the NAGLU gene, selection of Mut+, MutS and multiple integrant strains, and growth at decreased temperature were explored to optimise NAGLU expression in the methylotrophic yeast, Pichia pastoris. As none of these measures resulted in abundant NAGLU production, Sf9 and Tni insect cell lines were investigated as an alternate expression system. Additionally, a protein transduction domain (PTD) was fused to NAGLU (NTAT) to circumvent current problems faced in delivering therapeutic enzymes to the brain. NAGLU protein, with and without a fused PTD, were expressed using stable transfection and baculovirus infection techniques. Small scale experiments utilizing the baculovirus expression vector system (BEVS) have yielded promising results, generating functionally active NAGLU and NTAT protein of the expected approximately 80-85 kDa molecular mass. This preliminary success indicates the BEVS may be an attractive option for the large scale production of rNAGLU and rNTAT.

No description available.

Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive disorder caused by a defect in alpha-N-acetylglucosaminidase (NAGLU), a lysosomal enzyme involved in the degradation of heparan sulphate. Dysfunctional NAGLU gives rise to a clinical phenotype of severe and progressive mental retardation, often accompanied by hyperactivity and aggressive behaviour. At present, there is no effective treatment for MPS IIIB. However, cloning of the human NAGLU cDNA has made the potential production of human recombinant enzyme for use in enzyme replacement therapy (ERT) a viable option. The work outlined herein focuses on attempts to produce human recombinant NAGLU (rNAGLU) using both yeast and insect cell based expression systems; with the major focus on yeast based expression. Use of a humanized yeast strain, codon optimisation of a portion of the NAGLU gene, selection of Mut+, MutS and multiple integrant strains, and growth at decreased temperature were explored to optimise NAGLU expression in the methylotrophic yeast, Pichia pastoris. As none of these measures resulted in abundant NAGLU production, Sf9 and Tni insect cell lines were investigated as an alternate expression system. Additionally, a protein transduction domain (PTD) was fused to NAGLU (NTAT) to circumvent current problems faced in delivering therapeutic enzymes to the brain. NAGLU protein, with and without a fused PTD, were expressed using stable transfection and baculovirus infection techniques. Small scale experiments utilizing the baculovirus expression vector system (BEVS) have yielded promising results, generating functionally active NAGLU and NTAT protein of the expected approximately 80-85 kDa molecular mass. This preliminary success indicates the BEVS may be an attractive option for the large scale production of rNAGLU and rNTAT.